Glycoside metabolites of oospora virescens(link)wallr fungus

ABSTRACT

EIGHT GLYCOSIDE METABOLITE COMPOUNDS ARE PRODUCED BY THE OOSPORA VIRESCENS (LINK) WALLR FUNGUS IN GLUCOSEORGANIC DECOCTION MEDIA WHICH CAN BE SEPARATED FROM THE GROWTH MEDIA AND FROM EACH OTHER. THESE COMPOUNDS ARE EFFECTIVE BACTERIAL AND ANTIMYCOTIC AGENTS.

AP?il 2, 1974 N. c; BELLAVITA 3,801,565

GLYCOSIDE METABQLITES 0F OOSPORA VIRESCENS (LINK) WALLR FUNGUS FiledJan. 11, l97l 5 Sheets-Sheet 2 3000 I800 WAVENUMBER (CM ALISNHG "IVOIldOALISNBO IVOIidO April 2, 1974 Filed Jan. 11, 1971 INFRARED SPECTRUMOFIIVQB -(KBH l I400 I200 l l I 2500 2000 I800 WAVENUMBER l O O O O (Daouvwwsuvai ALISNBG wqudo N. c. BELLAVITA GLYCOSIDE METABOLITES 0FOOSPORA VIRESCENS (LINK) WALLR FUNGUS (cm-U 0F, A- (Ker) INFRARED 5Sheets-Sheet SPECTRUM aouviuwsNvai MJSNBO 'IVUIidO 2500 4 2000 4800WAVENUMBER April 2, 1974 N c. BELLAVITA 3,801,565

GLYCOSIDB METABOLITES OF OOSPORA VIRESCENS (LINK) WALLR FUNGUS FiledJan. 11, 1971 5 Sheets-Sheet 5 I II O O O 0 THIN LAYER CH ROMATOGRAMUnited States Patent Oflice 3,801,565

Patented Apr. 2, 1974 1 a 2 wherein R has the meaning given above and Yis CHO 3,801,565 or CH OH with the proviso that when Y is CH OH,GLYCOSIDE METABOLITES F OOSPORA R is hydrogen, and

VIRESCENS (LINK) WALLR FUNGUS Nera Cagnoli Bellavita, Via XX Settembre2,

Perugia, Italy Filed Jan. 11, 1971, Ser. No. 105,477 Int. Cl. C07c 47/18US. Cl. 260-210 R 15 Claims ABSTRACT OF THE DISCLOSURE Eight glycosidemetabolite compounds are produced by the Oaspora virescens (link) wallrfungus in glucoseorganic decoction media which can be separated from thegrowth media and from each other. These compounds CHBOH are effectivebacterial and antimycotic agents. H0 |--0 H I l H 03 & BACKGROUND OFTHEINVENTION H 0H The Oospora virescens '(link) wallr fungus formsglycoside metabolites. Two of these glycosides have been described in*Gazzetta Chimica Italiana, vol. 98, pp. 1354-1369, 1969.

SUMMARY OF THE INVENTION The mixture of metabolites produced in themedia can It "has now been discovered that under proper condij beseparated from the culture by lyophilizing the culture tions this funguscan produce six other glycoside metabo- "and extracting with ethanol,and separated from each lites as well using suitable culture media. Thenew com other by chromatographic column techniques according pounds ofthe present invention have the formulas, to the processes described morefully hereinbelow. In

addition, certain of the glycoside metabolites can be prepared fromothers of these compounds. The mixture of metabolites as well as theindividual glycosides are useful antimycotic agents. In addition,various of the glycoside compounds are useful antibacterial agents.

Therefore it is a principal object of the present invention to providenew compositions of matter and methods of preparing themr It is anotherobject to provide a mixture of glycosides COOH 0-CH: produced by theOaspora virescens '(link) wallr fungus. H 4-0 It is still another objectto provide a method of separating such glycosides from each other. It isanother object to provide a method of inhibiting OH the growth of fungi.

on It is another object to provide a method of inhibiting v p the growthof bacteria.

' Further objects will become apparent from the followwh r i R can behydrogen or y y ing detailed description thereof.

BRIEF DESCRIPTION OF THE DRAWINGS p 1 FIGS. 1-8 are infrared spectra ofcompounds I through a VIII. FIG. 9 is a picture of two developed thinlayer i chromatograms of compounds I through VIII.

a] Roux) 7 DETAILED DESCRIPTION OF THE INVENTION Y g H a Eight glycosidemetabolites can be obtained from suit- 7 1 O v able culture media of thefungus Oaspora virescens 7 (Link) wallr by lyophilization and ethanolicextraction of H r I the medium. The glycoside metabolites can then besepf arated from each other by chromatographic column. For

convenience, the eight compounds have been numbered g 'l l I throughVIII in the order of their-separation on a thin Compound I \H CH0 0 g oH 1 h H Compound II CHrOH /C H l 0 H OK i Compound III Q Compound V GHOH OH:

Com ound VI According to the present process, a glycoside-producingstrain of Oospora virescens (link) wallr in an aqueous glucose-organicdecoction media is cultivated at a temperature from 10 to 25 C. in astationary culture for a period of at least ten days.

The glycoside-producing starin of the Oospora virescens (link) wallr aredeuteromycetes of the Moniliaceae family, isolated from the deadbranches of the mulberry bush. A colony is selected in agar malt from acolony differentiated from others by a more rapid 'and intensesporulation. In organic solid or liquid media based upon malt and onvegetable decoctions with or without glucose, such as wheat kernels,corn kernels, oat kernels, carrots, kidney bean seeds and the like, thefungus colonies are round with a slightly sinuous outline, at firstfiatted and white, then becoming raised; they change with the degree ofdevelopment to become green. They become dark green and velvety withtime. The vegetative mycelium consists of hyaline, branched, septatehyphae, about 2.5 microns wide which support numerous conidiophoresbearing abundant catenulate spores, produced in basipetal succession invery long, flexible chains. The conidiophores are simple, hyaline, erector bent, about 30 to 40 microns long, slightly swollen at the base andarrowed towards the tip. The spores are fusiform, smooth, about 7 to 8microns long and about 2.5 to 3 microns wide, having a green or olivegreen color.

The temperature of cultivation can be from 10 to C., preferably from 23to 25 C. Growth of the fungus is slow at 10 C., and above about 25 C. nogrowth occurs.

The culture must be stationary in order for the glycosides of theinvention to be formed.

The organic media found suitable for the production of the glycosides ofthe invention includes aqueous decoctions of malt, glucose-yeast,glucose-carrot, glucose-wheat kernels, glucose-corn kernels,glucose-kidney bean seeds, glucose-pea seeds and glucose-lentil seeds.The preferred decoction is glucose-carrot.

The pH of the media is important, particularly with respect to theformation of Compounds I and IV. Preferably the pH of the culture mediais basic, most preferably within the range 8.6 to 8.8. This can be doneby adding a suitable base, such as an alkali metal or ammonium hy- Thecultures as prepared above are lyophilized and extracted with ethanol.The ethanol extract is then cooled slowly, when the metal salts of acidCompounds VII and VIII form a precipitate. The resultant mixture isfiltered and the filtrate dried and charged to a chromatographic columnto separate the remaining compounds from each other, using conventionaltechniques.

The compounds are best separated using a mixture of chloroform-methanol,but other known solvents can also be employed. The chromatogram isdeveloped in conventional manner using sulphuric acid.

The precipitated salts are treated with a suitable acid, such as dilutehydrochloric acid, and separated from each other on a chromatographiccolumn using chloroformmethanol as above.

In addition to the procedures described above, certain of the glycosidemetabolites described herein can also be prepared from others of thesecompounds. For example, Compound I can be prepared from Compound V bytritylation, acetylation, detritylation, oxido-elimination with theDoerings reagent, and subsequent deacetylation with sodium methoxide inmethanol. Compound I can also be prepared by reducing the Compound VIIwith a reducing agent, such as lithium aluminum hydride, to form theCompound V and treating as above to form Compound I.

The Compound IV can be prepared by a similar manner from the Compound VIand by similar reduction of Compound VIII.

The compounds of the invention show a wide spectrum of activity againstparasitic fungi, both of human beings and animals, in vitro and in vivo.In particular the Compounds I and IV are of interest in that they havedemonstrated very low toxicity in mice, the LD of I in mice was 250mg./kg. subcutaneous, the LD of IV in mice was 250 mg./kg. subcutaneous.The LD of ethanolic extract was 500 mg./kg. subcutaneous. The compoundsof the invention can be administered in the form of a powder, tablet,tincture or ointment containing one or more the compounds of theinvention as active ingredient, together with conventional fillers andcarriers. Suitable ointments can be prepared, for example, withexcipient lanolin or fatty acids, such as undecylic acid or caprylicacid as the carrier. Powders and tablets can also be prepared inconventional manner. For example, a typical tablet can contain 70 mg. ofthe active compound of the invention, 25 mg. of ascorbic acid and 3 mg.of potassium metabisulfite.

Several of the compounds of the invention are particularly effectiveagainst particular fungi. For example, Compound I is effective againstcutaneous mycosis from dermatophytes such as alopecia, eczema, herpes,ringworm, favus and histoplasmosis. Compound IV is of particularinterest against infections such as candidiasis or moniliasis andcryptococcosis (above all Cryptococcus neoformans). The latter compoundsare most particularly of interest since they are effective against thepathogenic yeast Candida albicans, also known asMonilia albicans, whichhas resisted all treatment to date. These compounds are also effectiveto inhibit the growth of bacteria, both of the Gram-positive and theGram-negative varieties.

The invention will be further illustrated by the following examples, butit is to be understood that the invention is not meant to be limited tothe details described therein. In the examples, all percentages are byWeight.

In preparing the cultures, the growth media were charged to one literRoux bottles and sterilized at a pressure of 24 p.s.i. for 3 to 4minutes and then inoculated with 2 ml. of a sterile, aqueous suspensionof abundant spores prepared from well sporulated, 20 to 40 day-oldcultures of Oospora virescens (link) wallr on oat-agar slants. Thefungus is grown in stationary culture in daylight, maintaining thetemperature from 23 to 25 C.

EXAMPLE 1 Three portions each of glucose-carrot and glucose-corn kernelsmedia after sterilization were adjusted to pH 5, 7 and 8.6 respectively,then inoculated and cultivated. After incubation for 20 to 30 days, themedia having pH 5 were found to have assimilated glucose slowly and thepH increased toward neutral values, whereas the asssimilation of glucosein the other portions was much more rapid and the reaction mediumincreased toward pH 9.

After incubation of the media having pH 8.6 for fifteen days, CompoundsII, V, VI, VII and VIII were produced; after 30 days, Compounds 1, IIIand IV were also produced. Maximum amounts of Compounds I and IV wereproduced in the glucose-carrot decoction having pH 8.6-8.8.

EXAMPLE 2 Production of the Compounds I to VIII was compared in avariety of cultivation media as follows:

, H (l) Malt extract (powder), 15 gm./l p 6 (2) Glucose (2%) yeastextract, 3 gm./l 7.8 (3) Glucose (2%) carrot 5.5 (4) Glucose (2%) wheatkernels 5.8 (5) Glucose (2%) corn kernels 5.5 (6) Glucose (2%) kidneybean seeds 6 (7) Glucose (2%) pea seeds 5.8 (8) Glucose (2%) lentilseeds 6 (9) Glucose (2%) potato 5.5

EXAMPLE 3 This example demonstrates that other media tested will notproduce the compounds of the invention and is given for purposes ofcomparison,

(1) Czapeks modified liquid, prepared by admixing 1 gram potassiumhydrogen phosphate, 0.5 gram magnesium sulfate (MgSO -7H O), 0.5 grampotassium chloride, 0.01 gram ferrous sulfate, 2 grams sodium nitrate,20 grams glucose and adding deionized water 7 to make up 1 liter involume. This media has a pH of 6-6.5.

(2) Veindelings liquid, prepared by admixing 2 grams NH OCO(CHOH) COON=H1 gram potassium dihydrogen phosphate, 1 gram MgsQyH O, 25 grams glucoseand deionized water to make up 1 liter in volume. This media had a pH of4.5-5.

(3) Carrot decoction (260 gms./l.), pH 6.

(4) Wheat kernel decoction (140 gms./l.), pH 6.

(5) Corn kernel decoction (200 gms./l.), pH 6.4.

(6) Kidney bean seed decoction (100 gms./ 1.), pH 6.2.

(7) Pea seed decoction (100 gms./l.), pH 6.

' (8) Lentil seed decoction (100 gms./l.), pH 6.

(9) Potato decoction (200 gms./l.), pH 5.5.

The above media were inoculated and cultivated as in Example 1.

The fungus grew poorly in media (1) and (2), but no sporulation wasnoted and none of the Compounds I to VIII were produced.

The fungus grew more or less abundantly and sporulation was good tomoderate for media (3) to (9); however, none of the Compounds I to VIIIwere produced after 30 days.

EXAMPLE 4 A medium for growth of Oospora virescens (Link) wallr funguscultures was prepared with carrot decoction in deionized water (250gms./l.) and sterilized at a temperature of about 120 C. for 2-3minutes. The decoction was filtered through cotton wool, glucose added,2% of the total, and 300 ml. of the resultant medium charged to threeliter Lepin bottles sterilized at a temperature of 120 C. for 3-4minutes. After cooling, the medium was adjusted to pH 8.6-8.8 withsterile sodium hydroxide solution (1 N) and inoculated with 5 m1. of asterile aqueous suspension of abundant spores. Cultivation of the sporeswas carried out following the procedure of Example 1.

After days incubation, Compounds II, V, VI, VII and VIII were produced;after 30 days, Compounds I, III and IV were also produced.

Three liters of lyophilized culture prepared as above were fullyextracted with ethanol in a Soxhlet extractor and concentrated to /3volume. The extract was cooled slowly, when 5 grams of precipitateformed, which included the sodium salts of Compounds VII and VIII. Themixture was filtered and the filtrate evaporated to dryness undervacuum. Twelve grams of product was obtained. The product was charged toa silica gel chromatographic column and eluted with a mixture of 93:7chloroform: methanol. The following compounds were progressivelyseparated and recovered:

Compound I, 250 mg.: T.L.C. 1 R, 0.65 (purple spot).-- This compound hasthe following properties: molecular formula, C 'H O [a] =123 (C. =0.96,methanol); M+=446 (molecular ions); ultraviolet spectrum has absorptionmaxima at 258 m (e=6000 ethanol). Its infrared spectra (presented asFIG. 1) shows the following peaks at wavelengths expresesd in reciprocalcentimeters when run in a KBr disc: 3401, 3077, 2959, 2924, 2899, 2874,2833, 2817, 1681, 1639, 1471, 1460, 1439, 1429, 1383, 1370, 1344, 1325,1292, 1274, 1221, 1178, 1138, 1091, 1047, 1015, 1000, 961, 952, 930,909, 893, 885, 868, 856, 826, 813.

This compound was soluble in chloroform, methanol, ethanol and ethylacetate. It forms an amorphous precipitate from solution in ethylacetate.

Compound II, 50 mg.: T.L.C. R, 0.52 (red spot).- This compound has thefollowing properties: molecular formula, C26H40O7; [OL] =-71.4 (C.=0.98,methanol); melting point 160-162" C.; ultraviolet spectrum ,shows noabsorption maxima between 220-400 mu; its infrared spectra (presented asFIG. 2), shows the folowing peaks: 3584, 3401, 3086, 3058, 2976, 2950,2907, 2865, 2825, 2801, 1709, 1681, 1667, 1639, 1490, 1471,

1 See footnote (1) at bottom of column 8.

This compound is soluble in chloroform, methanol, ethanol and iscrystallizable from ethyl acetate.

Compound III, 300 mg.: T.L.C. R; 0.42 (purple spot) .This compound hasthe following properties: molecular formula, C H O [a] =149 (C.=0.72,methanol; M+ 448; melting point 170-172 C.; ultraviolet spectrum showsno absorption maxima between 220-400 mu; its infrared spectra (presentedas FIG. 3), shows the following peaks: 3534, 3448, 3086, 3040, 2959,2950, 2924, 2899, 2865, 2817, 1689, 1681, 1639, 1471, 1447, 1429, 1412,1381, 1337, 1206, 1186, 1124, 1084, 1058, 1047, 1026, 1000, 921, 811,790.

This compound is soluble in chloroform, methanol and ethanol and iscrystallizable from methanol.

Compound IV, 300 mg.: T.L.C. R, 0.36 (brown spot).-This compound has thefollowing properties: molecular formula, C H O [a] =113 (C.=0.97,methanol); M+ 462; ultraviolet spectrum has absorption maxima at 258 mu(e='6000 ethanol); its infrared spectra (presented as FIG. 4), shows thefollowing peaks: 3401, 3077, 2959, 2915, 2874, 2841, 2817, 1695, 1639,1449, 1431, 1414, 1387, 1372, 1342, 1179, 1149, 1089, 1058, 1000, 926,910, 885, 862, 826.

This compound is soluble in chloroform, methanol, ethanol and ethylacetate and forms an amorphous precipitate from a solution of ethylacetate/petroleum ether.

Compound V, 2000 mg.: T.L.C. R,=0.34 (brown spot) .This compound has thefollowing properties: molecular formula, C H O [a] =32.3 (C.=1.05,methanol); melting point at about 110 C.; ultraviolet spectrum shows noabsorption maxima between 220-400 mu; its infrared spectra (presented asFIG. 5) shows the following peaks: 3401, 3086, 2924, 2874, 2817, 1639,1447, 1425, 1412, 1383, 1372, 1330, 1294, 1285, 1267, 1215, 1143, 1138,1086, 1015, 1000, 966, 952, 909, 879, 856, 833, 830.

This compound is soluble in chloroform, methanoland ethanol and forms anamorphous precipitate from ethyl acetate.

Compound VI, 4000 mg.: T.L.C. R,=0.16 (green spot).This compound has thefollowing properties molecular formula, C H O [a] =42.7 (C.=1.03,methanol); melting point at about 130 C.; ultraviolet spectrum shows noabsorption maxima between 220-400 mu; its infrared spectra (presented asFIG. 6), shows the following peaks: 3401, 3086, 2959, 2907, 2825, 1637,1451, 1443, 1425, 1414, 1383, 1372, 1156, 1138, 1070, 1010, 966, 941,934, 909', 883, 832, 829, 782.

This compound was soluble in methanol, ethanol and chloroform and formsan amorphous precipitate from acetone and ethyl acetate.

Five grams of the sodium salts reserved were dissolved in 200 ml. ofwater and added with 1 N hydrochloric acid up to pH 3. The resultantprecipitate was centrifuged and dried under vacuum. Three grams ofproducts obtained were charged to a chromatographic column containinggrams of silicic acid:celite (3:1) and eluted with chloroform:methanol(90: 10). Two additional compounds were separated and recovered.

Compound VII, 1400 mg.: T.L.C. R,=0.43 (brown spot).-This compound hasthe following properties: molecular formula, C H O [a] =85.2 (C.=1.1,methanol; melting point 192-194 C.; ultraviolet spectrum shows noabsorption maxima between 220-400 mu; infrared spectra of itscarboxymethyl ester (presented as FIG. 7), shows the following peaks:3425, 3086, 3003,

1 Thin layer chromatography (Kieselgel H Fluke.) activated for 60minutes at and eluted with chloroform-methanol mixture (85:15)detection: sulphuric acid 50% at 110 for 5 minutes.

2 Thin layer chromatography (Kieselgel H. Fluka) activated for 60minutes at 110 and eluted with chl01'oform-1nethano1- acetic acidmixture (80 15 5) detection Sulphuric acid 50% at 110 for 5 minutes.

This compound is soluble in chloroform, methanol and ethanol and formsan amorphous precipitate from ethanol.

Compound VIII, 2000 mg.: T.L.C. R =0.1 8 (green spot).This compound hasthe following properties: molecular formula, C H O [a] =82.3 (C.=1.13,methanol); melting point 188-l90 C.; ultraviolet spectrum shows noabsorption maxima between 220-400 mu; its infrared spectra (presented asFIG. '8) shows the following peaks: 3546, 3401, 3086, 2967, 2950, 2899,2865,

EXAMPLE 8 v "The. procedure of Example 6 was repeated exceptsubstituting one gram of Compound VIH. 400. mg. of Compound IV wereobtained.

" EXAMPLE 9 The compounds prepared as in Example 4 were tested asantimycotic agents at various concentration, in vitro onglucose-Sabourauds liquid containing 0.5/ chloroamphenicol. The tablebelow shows the minimum concentration in mcg./mK of test compound whichwas effective to inhibit the growth of test fungus.

TABLE I Minimum inhibitory concentration in mcg./ml.

VI V II I IV VIII VII III Aspergillusfumigatus 50 50 12.5 25 50 50 100Aaperaillusflapus 100 100 12. 100 50 100 Mucm: corymbzjgrn 50 50 50 12.5100 100 53 Pem'czllmm murneflei 12.5 25 50 12.5 25 100 50 50Epzdermophutonfloccosum. 50 8.12 25 25 50 100 50 100 Mlf08120l"LL7nCGfl18. 12.5 3.12 25 25 50 50 50 50 Tricho hutoqt rubrum- 2512.5 25 12.5 25 100 25 50 Candzaalb1cans 100 100 100 50 6.25 100 50 100Cryptococcus neoformans.. 0.18 0.78 0.09 6.25 0.04 1 56 0.18 50Hiatoplarma capaulatum 12.5 50

2833, 2817, 1730, 1667,1639, 1471, 1451, 1414, 1389, EXAMPLE Thiscompound is soluble in chloroform, methanol and ethanol and forms anamorphous precipitate from ethanol.

EXAMPLE 5 This example demonstrates the preparation of Compound I fromCompound V.

One gram of Compound V was stirred in one gram of triphenylchloromethaneand 4 ml. of pyridine for 72 hours at room temperature. The mixture wascooled to 0 C., and 6 ml. of pyridine and 6 ml. of acetic anhydrideadded. After standing for 24 hours at room temperature, the mixture waspoured into ice water. The precipitate was collected and charged to achromatographic column of silica gel. The product was eluted withbenzenezethyl acetate (90:10). The solvent was evaporated and 2.2 gramsof product were obtained. The product was then treated with acetic acid(80%) at 110 C. to form the tetra-acetyl derivative. 1.2 grams ofproduct were obtained after chromatographic separation and treated withDoerings reagent (sO -pyridine-DMSO-triethylamine) to give thecorresponding triacetyl derivative of I (0.9 gram). This compound wastreated with 2 ml. of 1% sodium methoxide in methanol for minutes at 60C. and allowed to stand for 45 minutes at room temperature. 500 mg. ofCompound I were obtained.

EXAMPLE 6 This example demonstrates the preparation of Compound V fromCompound VII- One gram of Compound VII was dissolved in tetrahydrofuranand added to one gram of LiAlH, over a one hour period at roomtemperature. The product was collected and separated on chromatographiccolumn. One gram of Compound V was obtained.

When the procedure of Example 5 was repeated, 500 mg. of Compound I wereobtained.

EXAMPLE 7 The procedure of Example 5 was followed except substitutingone gram of Compound VI. 400 mg. of Compound IV were obtained.

2 Thin layer chromatography (Kieselgel H Fluka) activated for 60 minutesat 110 and eluted with chloroform-methanolacetic acid mixture (80: 15:5)detection: Sulphuric acid 50% at 110 for 5 minutes.

Compounds I "and were tested as antibacterial agents. The minimuminhibitory concentration in mcg./ ml. are given in the table below.

TABLE II MIC =Minlmum inhibitory concentration.

I claim: 1. A compound selected from the group consisting of compoundshaving the formula E if? 15-; 4

wherein R can be hydrogen or hydroxyl,

, 1 12 I 4 5. A compound according to claim 1 of the formula or CH OHwith the proviso that R is hydrogen, and

wherein R has the meaning given'above and Y is CHO when Y is CH O'H,

HO/ ela I CHO CHZOH o-cH, 0 H H 11 H0 I )l 15 1-1 H H HO OH\ H H H OH Hv 6. A compound according to claim 1 of the formula 2. A compoundaccording to claim 1 of the formula l H0 H l 11 0-0 1 f .10 Q, o

1 H HO onion 0 l--| H OH H . I 40 v 3. A compound according to claim 1of the formula V5 OCH2 (II-[NEH o Hf H HO E 00011 0-011, OH H H 8. Analkali metal or ammonium salt of the compound 1 of claim 3. H0 H 9.Thesodium salt of the compound of claim 3.

kil 10. An alkali metal or ammonium salt of the com- H pound of claim 4.

11. The sodium salt of the compound of claim 4.

12. A method for the semisynthetic production of a 4. A compoundaccording to claim 1 of the formula compound of the formula startingfrom a compound of the formula coon on,

13. A method for the semisynthetic production of a compound of theformula CH /0 i 0 o H IHO 'I (Le l CHIOH l n no) comprising the stepsof: tritylating with triphenylchloromethane in pyridine; acetylatingwith acetic anhydride in pyridine; detritylating with acetic acid;oxido-eliminating with Doerings reagent and deacetylating in thepresence of a strong base.

14. A method for the semisynthetic production of a compound of theformula ;comprising the steps of: reducing said latter compound withLiAlH; in ether; tritylating with triphynylchlorometharie in pyridine;acetylating with acetic anhydride in pyridine; detritylating with aceticacid; oxide-eliminating starting from a compound of the formula HO COOHH HO OH (L with Doerings reagent; and deacetylating in the presence of astrong base.

j 15. A method for the semisynthetic production of a compound of theformula OHH starting from a compound of the formula CHgOH O-GH;

|-o H v H HO EL comprising the steps of tritylating withtriphenylchloromethane in pyridine; acetylating with acetic anhydride inpyridine; detritylating with acetic acid; oxido-eliminating withDoerings reagent; and deacetylating in the presence of a strong base.References Cited Bellavita et al.: Gazzetta Chimica Italiana," vol. 98,pp. 1354-1363.

JOHNNIE R. EROWN, Primary Examiner US. Cl. X.R.

